DeltaPsim Disruption Research Articles

Tanshinone IIA inhibits leukemia THP-1 cell growth by induction of apoptosis

Tanshinone IIA, a diterpene quinone extracted from the traditional herbal medicine, Salvia miltiorrhiza Bunge, has been reported to have anti-tumor effects on a large variety of cancer cells. The present study was undertaken to investigate the in vitro antiproliferation and apoptosis inducing effects of Tanshinone IIA on leukemia THP-1 cell lines and its mechanisms of action. MTT assay was used to detect the cell growth inhibitory rate; cell apoptotic rate and the mitochondrial membrane potential (Deltapsim) were investigated by flow cytometry (FCM), apoptotic morphology was observed by Hoechst 33258 staining and DNA fragmentation analysis. The expression of caspase-3 and different apoptosis modulators were analyzed by Western blotting. The results revealed that Tanshinone IIA inhibited the growth of THP-1 cells and caused significant apoptosis, the suppression was both in time- and dose-dependent manner. After treatment by Tanshinone IIA for 48 h, the percentage of disruption of Deltapsim gradually increased in a dose-dependent manner along with marked changes of cell apoptosis. Western blotting showed cleavage of the caspase-3 zymogen protein (32-kDa) with the appearance of its 20-kDa subunit and a dose-dependent cleavage of PARP, with the appearance of 89-kDa fragment; The expression of Bcl-2 and survivin was down-regulated remarkably while Bax expression was up-regulated concurrently after the cells were treated with Tanshinone IIA for 48 h. We therefore conclude that Tanshinone IIA has significant growth inhibition effects on THP-1 cells by induction of apoptosis, and that Tanshinone IIA-induced apoptosis on THP-1 cells is mainly related to the disruption of Deltapsim and activation of caspase-3 as well as down-regulation of anti-apoptotic protein Bcl-2, survivin and up-regulation of pro-apoptotic protein Bax. The results indicate that Tanshinone IIA may serve as a potential anti-leukemia reagent.

Sodium Selenite Induces Apoptosis in Acute Promyelocytic Leukemia-Derived NB4 Cells Through Mitochondria-Dependent Pathway

Our previous study has shown that sodium selenite can cause apoptosis in acute promyelocytic leukemia-derived NB4 cells in a caspase-dependent manner involving Deltapsim disruption and cleavage of Bcl-2, but more detailed mechanism(s) remain unclear. Here we showed that mitochondrial apoptosis signaling pathway played a vital role in apoptosis induced by sodium selenite based on the following findings: 1) cytochrome c release, activation of caspase 9, mitochondrial targeting, and oligermerization of Bax; 2) caspase 9, but not caspase 8, inhibitor could attenuate apoptosis; 3) downregulation of Bax and Bad by siRNA could delay sodium selenite-induced apoptosis. Further investigation showed that ROS was an essential inducer of deltapsim disruption and apoptosis by sodium selenite. Our findings here demonstrate that sodium selenite can induce apoptosis in NB4 cells through a mechanism involving ROS, activation of proapoptotic proteins Bad and Bax, Deltapsim disruption, release of cytochrome c, and consequent initiation of caspase cascade.

Neuroprotective Role of Antidiabetic Drug Metformin Against Apoptotic Cell Death in Primary Cortical Neurons

Oxidative damage has been reported to be involved in the pathogenesis of diabetic neuropathy and neurodegenerative diseases. Recent evidence suggests that the antidiabetic drug metformin prevents oxidative stress-related cellular death in non-neuronal cell lines. In this report, we point to the direct neuroprotective effect of metformin, using the etoposide-induced cell death model. The exposure of intact primary neurons to this cytotoxic insult induced permeability transition pore (PTP) opening, the dissipation of mitochondrial membrane potential (DeltaPsim), cytochrome c release, and subsequent death. More importantly, metformin, together with the PTP classical inhibitor cyclosporin A (CsA), strongly mitigated the activation of this apoptotic cascade. Furthermore, the general antioxidant N-acetyl-L: -cysteine also prevented etoposide-promoted neuronal death. In addition, metformin was shown to delay CsA-sensitive PTP opening in permeabilized neurons, as triggered by a calcium overload, probably through its mild inhibitory effect on the respiratory chain complex I. We conclude that (1) etoposide-induced neuronal death is partly attributable to PTP opening and the disruption of DeltaPsim, in association with the emergence of oxidative stress, and (2) metformin inhibits this PTP opening-driven commitment to death. We thus propose that metformin, beyond its antihyperglycemic role, can also function as a new therapeutic tool for diabetes-associated neurodegenerative disorders.

Induction of Apoptosis by Disturbing Mitochondrial-Membrane Potential and Cleaving PARP in Jurkat T Cells through Treatment with Acetoxyscirpenol Mycotoxins

Paecilomyces tenuipes is a famous Chinese medicinal entomopathogenic fungus that grows within the larvae of silkworms. 4beta-acetoxyscirpendiol (4-MAS), a cytotoxic compound belonging to the scirpenol subfamily of trichothecene mycotoxin, was isolated from Paecilomyces tenuipes. To further elucidate the cytotoxic mechanism of 4-MAS, evidences of its induction of apoptosis, together with the structurally related acetoxyscirpenol moiety mycotoxins (ASMs) such as, 15-acetoxyscirpenol (15-MAS), 4,15-diacetoxyscirpenol (4,15-DAS), and 3alpha-acetyldiacetoxyscirpenol (TAS), in the human Jurkat T cell line were reported herein. In the MTT reduction and time-course cytotoxicity assays for monitoring cell viability, all the four ASMs that were tested exhibited cytotoxicity; single acetoxylation at C-4 of the scirpenol family resulted in relatively weak cytotoxicity, while acetoxylation at C-15 resulted in strong cytotoxicity regardless of the other acetoxylations at the C-3 and/or C-4 positions. Phosphatidylserine externalization was induced by all the ASMs that were treated at an early phase in a time-dependent manner, showing a typical apoptotic phenomenon, not a necrotic one. The ASMs also reduced the mitochondria's inner-membrane potential (deltaPsim) through flow cytometry analysis after staining these with DiOC6, a mitochondria-specific and voltage-dependent dye. Acetoxylation of ASM at C-15 increased deltaPsim disruption, but that at C-3 reduced the deltaPsim. The ASMs that were tested also cleaved 113 kDa PARP to an 89-kDa fragment through Western blot assay, suggesting the activation of caspase-3 and/or caspase-7 in the Jurkat T cell. DNA fragmentation was also observed to have been increased in a time-dependent manner by the ASMs that were tested in Jurkat T cells, resulting in the DNA fragmentation intensity order of 4,15-DAS>15-MAS>TAS>4-MAS. These data indicate that the Jurkat T cells that were treated with ASMs underwent typical cascades of apoptotic cell death.

TGF β1 induces prolonged mitochondrial ROS generation through decreased complex IV activity with senescent arrest in Mv1Lu cells

Transforming growth factor beta1 (TGF beta1) is a well-characterized cytokine that suppresses epithelial cell growth. We report here that TGF beta1 arrested lung epithelial Mv1Lu cells at G1 phase of the cell cycle with acquisition of senescent phenotypes in the presence of 10% serum, whereas it gradually induced apoptosis with lower concentrations of serum. The senescent arrest was accompanied by prolonged generation of reactive oxygen species (ROS) and persistent disruption of mitochondrial membrane potential (DeltaPsim). We demonstrated that the sustained ROS overproduction was derived from mitochondrial respiratory defect via decreased complex IV activity and was involved in the arrest. Moreover, we verified that hepatocyte growth factor released Mv1Lu cells from the arrest by protecting mitochondrial respiration, thereby preventing both the DeltaPsim disruption and the ROS generation. Our present results suggest the TGF beta1-induced senescent arrest as another plausible mechanism to suppress cellular growth in vivo and provide a new biochemical association between the mitochondrial functional defects and the cytokine-induced senescent arrest, emphasizing the importance of maintenance of mitochondrial function in cellular protection from the arrest.

Cloning and Identification of Hepatocellular Carcinoma Down-regulated Mitochondrial Carrier Protein, a Novel Liver-specific Uncoupling Protein

We report the identification of a novel cDNA fragment that shows significantly reduced expression in cancerous tissue compared with paired non-cancerous liver tissue in patients with hepatocellular carcinoma (HCC). The full-length transcript of 1733 bp encodes a protein of 308 amino acids that has all the hallmark features of mitochondrial carrier proteins. We designate the novel protein as HDMCP (HCC-down-regulated mitochondrial carrier protein). The HDMCP orthologs in human, mouse, and rat are found to exhibit close similarity in protein sequence and gene organization, as well as exclusive expression in the liver. Moreover, conserved syntenic regions have been demonstrated at the HDMCP gene locus in the human, mouse, and rat genome. Taken together, we suggest that HDMCP might have a conserved and unique biological function in the liver. Overexpression of HDMCP in transiently transfected cancer cells results in the loss of staining by MitoTracker dye, indicating that HDMCP could induce the dissipation of mitochondrial membrane potential (DeltaPsim). However, HDMCP-mediated disruption of DeltaPsim is not related to mitochondrial permeability transition or apoptosis. In addition, we further demonstrate that the dissipation of DeltaPsim is accompanied by significant reduction of cellular ATP in 293T cells overexpressing HDMCP or uncoupling protein 2 (UCP2). Our present findings suggest that HDMCP might be one of the long postulated uncoupling proteins that catalyze the physiological "proton leak" in the liver. The down-regulation of HDMCP in HCC cancer cells might result in the elevation of DeltaPsim, a common phenomenon found in cancer cells.

Mitochondrial alterations precede Bordetella pertussis-induced apoptosis.

Bordetella pertussis, the causative agent of whooping cough in humans, secretes a number of toxins, including adenylate cyclase-hemolysin (AC-Hly), and induces macrophage apoptosis. We investigated the effects of B. pertussis on mitochondrial membrane potential (deltapsim) and ATP levels, as possible determinants of cell death. Using the fluorescent probe JC-1, we found that infection of human monocytes by B. pertussis lead to a disruption in host-cell deltapsim. deltapsim alterations were preceded by a massive increase in cyclic AMP, a moderate decrease in ATP, and was independent from oxidative stress. These changes were observed when human monocytes were infected by the parental B. pertussis 18323 but not when infected by the mutants deficient in the expression of AC-Hly. Exposure of human monocytes to purified AC-Hly induced changes comparable to those observed with the B. pertussis parental strain. Our results provide a mechanistic relationship between AC-Hly, ATP, and deltapsim disruption in the cascade of events leading to B. pertussis-induced apoptosis.

Evidence for the induction of apoptosis by endosulfan in a human T-cell leukemic line.

Several organochlorinated pesticides including DDT, PCBs and dieldrin have been reported to cause immune suppression and increase susceptibility to infection in animals. Often this manifestation is accompanied by atrophy of major lymphoid organs. It has been suggested that increased apoptotic cell death leading to altered T-B cell ratios, and loss of regulatory cells in critical numbers leads to perturbations in immune function. The major objective of our study was to define the mechanism by which endosulfan, an organochlorinated pesticide, induces human T-cell death using Jurkat, a human T-cell leukemic cell line, as an in vitro model. We exposed Jurkat cells to varying concentrations of endosulfan for 0-48 h and analyzed biochemical and molecular features characteristic of T-cell apoptosis. Endosulfan lowered cell viability and inhibited cell growth in a dose- and time-dependent manner. DAPI staining was used to enumerate apoptotic cells and we observed that endosulfan at 10-200 microM induced a significant percentage of cells to undergo apoptotic cell death. At 48 h, more than 90% cells were apoptotic with 50 microM of endosulfan. We confirmed these observations using both DNA fragmentation and annexin-V binding assays. It is now widely being accepted that mitochondria undergo major changes early during the apoptotic process. We examined mitochondrial transmembrane potential (deltapsim) in endosulfan treated cells to understand the role of the mitochondria in T-cell apoptosis. Within 30 min of chemical exposure, a significant percentage of cells exhibited a decreased incorporation of DiOC6(3), a cationic lipophilic dye into mitochondria indicating the disruption of deltapsim. This drop in deltapsim was both dose- and time-dependent and correlated well with other parameters of apoptosis. We also examined whether this occurred by the down regulation of bcl-2 protein expression that is likely to increase the susceptibility of Jurkat cells to endosulfan toxicity. Paradoxically, the intracellular expression of bcl-2 protein was elevated in a dose dependent manner suggesting endosulfan-induced apoptosis occurred by a non-bcl-2 pathway. Based on these data, as well as those reported elsewhere, we propose the following sequence of events to account for T-cell apoptosis induced by endosulfan: uncoupling of oxidative phosphorylation --> excess ROS production --> GSH depletion --> oxidative stress --> disruption of deltapsim --> release of cytochrome C and other apoptosis related proteins to cytosol --> apoptosis. This study reports for the first time that endosulfan can induce apoptosis in a human T-cell leukemic cell line which may have direct relevance to loss of T cells and thymocytes in vivo. Furthermore, our data strongly support a role of mitochondrial dysfunction and oxidative stress in endosulfan toxicity.

Susceptibility of HIV-1-TAT transfected cells to undergo apoptosis. Biochemical mechanisms.

The effects of HIV-1 Tat protein on mitochondria membrane permeability and apoptosis were analysed in lymphoid cells. In this report we show that stable-transfected HIV-Tat cells are primed to undergo apoptosis upon serum withdrawal. This effect was observed in both the Jhan T cell line and the K562 cells, the latter expressing the bcr-abl chimeric gene, which confers resistance to apoptosis induced by different stimuli. Using a cytofluorimetric approach we have determined that serum withdrawal induces a disruption of the transmembrane mitochondrial potential (Deltapsim) followed by an increase of reactive oxygen species (ROS) and the subsequent DNA nuclear loss in K562-Tat cells but not in the K562-pcDNA cell line. These pre-apoptotic events were associated with the cleavage of the caspase-3, while the expression of Bcl-2, Bcl-XL and Bax proteins was not affected by the presence of Tat. Regardless of the steady state of the Bax protein, we found that in both K562 and K562-Tat cells, this protein is located in the nucleus, but after serum withdrawal its localization was mainly in the cytoplasm. The activity of caspase-3 detected in K562-Tat cells after serum withdrawal paralleled with the mitochondria permeability transition. Nevertheless, in Jhan-Tat cells the inhibition of this caspase with the specific inhibitor, z-DEVD-cmk, did not affect the disruption of the mitochondria potential induced by serum withdrawal. Interestingly, we found that HIV-Tat protein accumulates at the mitochondria in the K562-Tat cells cultured under low serum conditions, and this mitochondrial localization correlated with the Deltapsim disruption detected in these cells. In addition, HIV-1 Tat protein synergies with protoporphyrin IX (PPIX), a ligand of the mitochondrial benzodiazepine receptor, in the induction of apoptosis in both Jhan and K562 cells. Thus, HIV-1 Tat protein may induce apoptosis by a mechanism that involves mitochondrial PT and may contribute to the lymphocyte depletion seen in AIDS patients.